Human umbilical cord cells for cardiovascular tissue engineering: a comparative study.

OBJECTIVE: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering. METHODS: Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing. RESULTS: UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs. CONCLUSIONS: Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.     

A new source for cardiovascular tissue engineering: human bone marrow stromal cells

Objective: Vascular-derived cells represent an established cell source for tissue engineering of cardiovascular constructs. Previously, cell isolation was performed by harvesting of vascular structures prior to scaffold seeding. Marrow stromal cells (MSC) demonstrate the ability to differentiate into multiple mesenchymal cell lineages and would offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. We studied the feasibility of using MSC as an alternative cell source for cardiovascular tissue engineering. Methods: Human MSC were isolated from bone marrow and expanded in culture. Subsequently MSC were seeded on bioabsorbable polymers and grown in vitro. Cultivated cells and seeded polymers were studied for cell characterization and tissue formation including extracellular matrix production. Applied methods comprised flow cytometry, histology, immunohistochemistry, transmission (TEM) and scanning electron microscopy (SEM), and biochemical assays. Results: Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for alpha-smooth muscle actin and vimentin. Histology and SEM of seeded polymers showed layered tissue formation. TEM demonstrated formation of extracellular matrix with deposition of collagen fibrils. Matrix protein analysis showed production of collagen I and III. In comparison to vascular-derived cell constructs quantitative analysis demonstrated comparable amounts of extracellular matrix proteins in the tissue engineered constructs. Conclusions: Isolated MSC demonstrated myofibroblast-like characteristics. Tissue formation on bioabsorbable scaffolds was feasible with extracellular matrix production comparable to vascular-cell derived tissue engineered constructs. It appears that MSC represent a promising cell source for cardiovascular tissue engineering.     

冷冻保存人脐带血管细胞培育组织工程心血管补片

目的 探讨应用液氮冷冻保存的人脐带动脉壁肌成纤维细胞(HUAMs)制备组织工程心血管补片的可行性.方法 将液氮冷冻保存的人脐带动脉壁肌成纤维细胞种植在聚-4-羟基丁酸酯(P4HB)聚合材料构建的补片支架材料上,制备组织工程心血管补片.体外静态培育21 d,对补片组织进行组织学、扫描电镜检查.免疫组织化学分析组织样本中细胞外基质(胶原蛋白)的合成.结果 苏木素-伊红(HE)染色显示,肌成纤维细胞较好地黏附于聚合材料上,大部分细胞保持生长活力,并浸润生长入支架材料内部,形成组织.免疫组织化学显示补片材料中有胶原蛋白合成.结论 液氮冷冻保存的人类脐带肌成纤维细胞可作为种子细胞,用于制备人组织工程心血管补片.     

Stem cells used for cardiovascular tissue engineering

Stem cell research and tissue engineering have become leading fields in basic research worldwide. Especially in cardiovascular medicine, initial reports on the potential of using stem cells to recover cardiac function and replace organ subunits such as heart valves seemed to offer the promise of widespread clinical use in the near future. However, the broad application of this new therapy failed due to safety and efficacy concerns. Due in part to the initial reports, major basic research efforts were undertaken to explore the specific cell types in greater detail and identify their mechanisms of supporting function, resulting in remarkable new findings in stem cell biology. For example, the notion of resident human cardiac stem cells has disproved the earlier supposition that the human heart is a finitely differentiated organ without the intrinsic potential for regeneration. Furthermore, new technologies emerged to produce pluripotent cells without the ethical and immunological drawbacks of embryonic stem cells (for instance by nuclear transfer). Other autologous cell sources are presently under investigation in myocardial tissue engineering. For tissue engineering of heart valves and small calibre vessels, the use of autologous endothelial (precursor) cells may be the optimal means of seeding a biological or artificial scaffold. It is important that ongoing basic and clinical research in cardiovascular surgery might explore the potential of different cell types either using tissue engineering constructs or in cell transplantation approaches.     

Human dental pulp cells: a new source of cell therapy in a mouse model of compressive spinal cord injury

Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1?min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.     

磷酸钙骨水泥复合物/脐带间充质干细胞凝胶构建组织工程骨

目的构建新型可注射强化型磷酸钙骨水泥复合物/脐带间充质干细胞凝胶组织工程骨,探讨其力学性能,细胞活性和成骨作用。方法选用第四代hUCMSCs,1.2%海藻酸钠水凝胶构建hUCMSCs水凝胶微球。高温煅烧钙/磷比约为1.9的磷酸氢钙和碳酸钙混合物,按摩尔质量比以1：1混合制备CPC粉末。15%Chitosan,8mm长度可吸收纤维用于提高CPC复合物力学强度。实验组分为四组：（1）单纯hUCMSCs微球;（2）CPC＋hUCMSCs微球;（3）CPC＋chitosan＋hUCMSCs微球;（4）CPC＋chitosan＋可吸收纤维＋hUCMSCs微球,分别检测力学性能,细胞活性和成骨作用。结果新型组织工程骨力学性能显著增强,抗弯曲强度提高到（11.7±2.1）MPa,弹性模量提高到（2.0±0.4）GPa,断裂功提高到（1.65±0.66）kj/m2（P〈0.05）。hUCMSCs的ALP活性第7天时明显增高,第14天时达峰,第21天时有所减弱,各组间比较无明显统计学差异（P〉0.1）。第7天时,所有实验组中的hUCMSCs均见少量矿物合成。第14天和第21天时,矿物合成数量明显增多。hUCMSCs中ALP基因表达培养第1天最低,第4天达峰,第8天时稍减弱。OC基因表达第8天达峰。结论构建完成新型可注射强化型磷酸钙骨水泥/脐带间充质干细胞凝胶构建组织工程骨。水凝胶微球中hUCMSCs在CPC中具有良好的成骨作用。CPC-chitosan-可吸收纤维组织工程骨力学性能满足松质骨力学要求,支持水凝胶微球中hUCMSCs的细胞活性和成骨作用。为组织工程骨研究和临床应用提供新思路和新方法。     

Selection of cell source for ligament tissue engineering

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as alpha-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and alpha-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.     

Human umbilical cord blood-derived stromal cells: a new resource in hematopoietic reconstitution in mouse haploidentical transplantation

Objective

Our previous study showed that human umbilical cord blood-derived stromal cells (hUcBdSCs) expanded CD34⁺ cells in vitro. This study further explored the role of hUcBdSCs in vivo.

Methods

The cultured hUcBdSCs were infused into transplanted haploidentical mice to observe hematopoietic recovery and complications.

Results

The engraftment was faster in transplantation with hUcBdSCs than without hUcBdSCs. The numbers of fibroblast (CFU-F), granulocyte/monocyte (CFU-GM), erythrocytic (CFU-E), and megakaryocyte (CFU-Mg) colony-forming units were greater among mice transplanted with hUcBdSCs than without hUcBdSCs. The scoring of graft-versus-host disease was significantly lower in mice that had been subjected to transplantation with hUcBdSCs than without hUcBdSCs. The infused hUcBdSCs migrated to the bone marrow of the recipients.

Conclusions

These data indicated that hUcBdSCs improved hematopoietic reconstitution in haploidentical transplantation in mice.     

Bone marrow as a cell source for tissue engineering heart valves

Background

This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach.

Methods

Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing.

Results

Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including α-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets.

Conclusions

Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form “tissue” that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.     

Application of stem cells for cardiovascular grafts tissue engineering

Congenital and acquired heart diseases are leading causes of morbidity and mortality world-wide. Currently, the synthetic materials or bioprosthetic replacement devices for cardiovascular surgery are imperfect and subject patients to one or more ongoing risks including thrombosis, limited durability and need for reoperations due to lack of growth in children and young adults. Suitable replacement grafts should have appropriate characteristics, including resistance to infection, low immunogenicity, good biocompatability and thromboresistance, with appropriate mechanical and physiological properties. Tissue engineering is a new scientific field aiming at fabrication of living, autologous grafts having structure or function properties that can be used to restore, maintain or improve tissue function. The use of autologous stem cells in cardiovascular tissue engineering is quite promising due to their capacity of self-renewal, high proliferation, and differentiation into specialized progeny. Progress has been made in engineering the various components of the cardiovascular system, including myocardial constructs, heart valves, and vascular patches or conduits with autologous stem cells. This paper will review the current achievements in stem cell-based cardiovascular grafts tissue engineering, with an emphasis on its clinical or possible clinical use in cardiovascular surgery.     

The placenta as a cell source in fetal tissue engineering

Purpose: This study was aimed at determining whether fetal tissue constructs can be engineered from cells derived from the placenta. Methods: A subpopulation of morphologically distinct cells was isolated mechanically from specimens of human placenta (n = 6) and selectively expanded. The lineage of these cells was determined by immunofluorescent staining against multiple intermediate filaments and surface antigens. Cell proliferation rates were determined by oxidation assays and compared with those of immunocytochemically identical cells derived from human amniotic fluid samples (n = 6). Statistical analysis was by analysis of variance (ANOVA). After expansion, the cells were seeded onto a polyglycolic acid polymer/poly-4-hydroxybutyrate scaffold. The resulting construct was analyzed by both optical and scanning electron microscopy. Results: The immunocytochemical profile of expanded placental cells was consistent with a nontrophoblastic, mesenchymal origin. Their proliferation rate in culture was not significantly different when compared with mesenchymal fetal cells isolated from human amniotic fluid; however, it was greater than previously reported rates for similar cells obtained from postnatal or adult tissues. Construct analysis showed dense layers of cells firmly attached to the scaffold without evidence of cell death. Conclusions: Subpopulations of nontrophoblastic, mesenchymal cells can be isolated consistently from the human placenta. These cells proliferate as rapidly as fetal mesenchymal amniocytes in vitro and attach firmly to polyglycolic acid scaffolds. The placenta can be a valuable and practical source of cells for the engineering of select fetal tissue constructs. J Pediatr Surg 37:995-999.     

人脐带血间充质干细胞研究近况及其在脂肪组织工程中的应用

人脐带血间充质干细胞(HUCBMSCs)是一种具有全能干细胞特点的细胞,是中胚层发育的早期细胞,其形态和生物学特点与骨髓源性间充质干细胞相似,具有多向分化潜能,可分化为成骨细胞、脂肪细胞、神经细胞等[1]。与骨髓相比,脐带血有更充足的来源,对供者无任何不良影响,其间充质干细胞更为原始,分化能力更强。因此脐带血间充质干细     

人脐带间充质干细胞与蚕丝蛋白支架构建组织工程脂肪的研究

目的：将体外以人脐带间充质干细胞（hUCMSCs）与蚕丝蛋白支架初步构建的组织工程脂肪移植到大鼠体内,观察其演变过程。方法：hUCMSCs与蚕丝蛋白支架复合培养10天后,进行成脂诱导;6周后将其移植到Wi st ar大鼠后肢肌肉内,同时,以同体积支架材料作为对照;分别于移植后4周和8周取材,行油红O染色、HE染色以及扫描电镜观察。结果：hUCMSCs与蚕丝蛋白支架复合培养及成脂诱导6周后,见大量成脂样细胞生成,并与支架牢固粘附。移植4周,移植物体积略小,质稍硬,表面有透明薄膜形成,膜中分布新生血管网;油红O染色见支架内新生脂肪组织及细胞呈橙红色;HE染色显示支架网眼内有新生脂肪组织,并可见少量炎性细胞浸润;扫描电镜见支架网眼内有球形、表面光滑的脂肪细胞。移植8周,移植物体积进一步缩小,质变软,表面薄膜内血管网丰富;油红O染色见支架中着橙红色组织较前明显增多,部分呈片状融合;HE染色显示新生脂肪明显增多,仍有少量炎性细胞浸润;扫描电镜显示脂肪细胞较前增生明显。对照组同样可见炎性细胞浸润,未见新生脂肪组织生成,支架材料8周时较4周时降解更加明显。结论：随着时间推移,蚕丝蛋白支架网眼内脂肪细胞逐渐增多,支架材料在体内呈现逐步降解趋势,说明体内环境有利于组织工程化脂肪的进一步形成。同时,也提示支架材料在组织相容性方面尚存不足。     

Cryopreservation of vascular mixed cell for tissue engineering in cardiovascular surgery]

Tissue engineering (TE) is a new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffold. Various vascular and valvular grafts have been tried to create with this TE approach. In clinical use of this technique, harvested and cultured cells have to keep viability until implantation as tissue engineered tissue. But few research for cryopreservation of vascular mixed cells has been performed. So, we investigated the proper method for cryopreservation of vascular mixed cells harvested from femoral artery and vein of dogs. Cells were cultured and divide into three groups, A: cryopreserving in 5% dimethylsulfoxide (DMSO), hydroxyethyl starch (HES), and fetal bovine serum (FBS) with -80 degrees C freezer; B: cryopreserving in 10% DMSO and FBS with programmed freezer; C: control (continuous culture in media). After rapid thawing at 40 degrees C, group A showed higher viability than group B with flow cytometry. The results means that vascular mixed cells can be successfully cryopreserved in the DMSO/HES mixture simply and inexpensively, without rate controlled freezing.     

Human umbilical cord blood mononuclear cells for the treatment of acute myocardial infarction

Cell transplantation is a new treatment to improve cardiac function in hearts that have been damaged by myocardial infarction. We have investigated the use of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction. The control group consisted of 24 normal rats with no interventions. The infarct + vehicle group consisted of 33 rats that underwent left anterior descending coronary artery (LAD) ligation and after 1 h were given Isolyte in the border of the infarction. The infarct + HUCBC group consisted of 38 rats that underwent LAD ligation and after 1 h were given 10(6) HUCBC in Isolyte directly into the infarct border. Immunosuppression was not given to any rat. Measurements of left ventricular (LV) ejection fraction, LV pressure, dP/dt, and infarct size were determined at baseline and 1, 2, 3, and 4 months. The ejection fraction in the controls decreased from 88+/-3% to 78+/-4% at 4 months (p = 0.03) as a result of normal aging. Following infarction in the infarct + vehicle group, the ejection fraction decreased from 87+/-4% to 51+/-3% between 1 and 4 months (p < 0.01). In contrast, the ejection fraction of the infarcted + HUCBC-treated rat hearts decreased from 87+/-4% to 63+/-3% at 1 month, but progressively increased to 69+/-6% at 3 and 4 months, which was different from infarct + vehicle group rats (p < 0.02) but similar to the controls. At 4 months, anteroseptal wall thickening in infarct + HUCBC group was 57.9+/-11.6%, which was nearly identical to the control anteroseptal thickening of 59.2+/-8.9%, but was significantly greater than the infarct + vehicle group, which was 27.8+/-7% (p < 0.02). dP/dt(max) increased by 130% in controls with 5.0 microg of phenylephrine (PE)/min (p < 0.001). In the infarct + vehicle group, dP/dt(max) increased by 91% with PE (p = 0.01). In contrast, in the infarct + HUCBC group, dP/dt(max) increased with PE by 182% (p < 0.001), which was significantly greater than the increase in dP/dt(max) in the infarct + vehicle group (p = 0.03) and similar to the increase in the controls. Infarct sizes in the infarct + HUCBC group were smaller than the infarct + vehicle group and averaged 3.0+/-2.8% for the infarct + HUCBC group versus 22.1+/-5.6% for infarct + vehicle group at 3 months (p < 0.01); at 4 months they averaged 9.2+/-2.0% for infarct + HUCBC group versus 40.0+/-9.2% for the infarct + vehicle group (p < 0.001). The present experiments demonstrate that HUCBC substantially reduce infarction size in rats without requirements for immunosuppression. As a consequence, LV function measurements, determined by LV ejection fraction, wall thickening, and dP/dt, are significantly greater than the same measurements in rats with untreated infarctions.     

Human umbilical cord mesenchymal stem cells co-culture ameliorates podocytic apoptosis: a possible role of HGF

Objective To explore the effects of human umbilical cord mesenchymal stem cells(HUC-MSCs) on podocytic apoptosis and injury induced by high glucose (HG) and the underlying mechanisms. Methods Podocytes were divided into six groups according to treatment: ⑴ normal glucose group (NG); ⑵ high glucose group (HG); ⑶ mannitol control group (NG+Ma); ⑷ HUC-MSC co-culture group (HUC-MSCs); ⑸ recombinant human hepatocyte growth factor treatment group (rhHGF); ⑹ neutralizing antibody group(HGF-NtAb). Cytometry and Hoechst staining were used to detect the apoptosis rates. Western blot was used to measure the ratio of active PARP to total PARP and the level of Bcl-2. Immunofluorescence was used to study podocytic apoptosis and injury. Neutralizing antibody (NtAb) was used to block its function and the recombinant cytokine was added to induce its function. Results High glucose induced podocytic apoptosis in a time-dependent manner, HUC-MSCs co-culture decreased the podocytic apoptosis rate and the expression of PARP (all P﹤0.05), increased the expression of Bcl-2, prevented the reduced expression and maintained the normal arrangement of podocytic podoplanin. The rhHGF prevented podocytic apoptosis and injury similarly to HUC-MSCs, the beneficial effect of HUC-MSC decreased when blockade of HGF. Conclusions HUC-MSCs co-culture ameliorates podocytic apoptosis and injure induced by HG, probably through secreting soluble HGF.     

Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering

We have isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a fibroblast-like morphology. However, this morphology changed after 5-azacytidine treatment in about 30% of the cells, which connected with adjoining cells after 1 week, formed myotube-like structures and began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. These cells expressed atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres and atrial granules. They had sinus node-like or ventricular cell-like action potentials. Analysis of the isoform of contractile protein genes, such as myosin and alpha-actin, indicated that their phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine treatment, and expressed MEF2-A and MEF2-D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte transplantation.     

Fibrin gel -- advantages of a new scaffold in cardiovascular tissue engineering.

OBJECTIVE: The field of tissue engineering deals with the creation of tissue structures based on patient cells. The scaffold plays a central role in the creation of 3-D structures in cardiovascular tissue engineering like small vessels or heart valve prosthesis. An ideal scaffold should have tissue-like mechanical properties and a complete immunologic integrity. As an alternative scaffold the use of fibrin gel was investigated. METHODS: Preliminary, the degradation of the fibrin gel was controlled by the supplementation of aprotinin to the culture medium. To prevent tissue from shrinking a mechanical fixation of the gel with 3-D microstructure culture plates and a chemical fixation with poly-L-lysine in different fixation techniques were studied. The thickness of the gel layer was changed from 1 to 3 mm. The tissue development was analysed by light, transmission and scanning electron microscopy. Collagen production was detected by the measurement of hydroxyproline. Injection molding techniques were designed for the formation of complex 3-D tissue structures. RESULTS: The best tissue development was observed at an aprotinin concentration of 20 microg per cc culture medium. The chemical border fixation of the gel by poly-L-lysine showed the best tissue development. Up to a thickness of 3 mm no nutrition problems were observed in the light and transmission electron microscopy. The molding of a simplified valve conduit was possible by the newly developed molding technique. CONCLUSION: Fibrin gel combines a number of important properties of an ideal scaffold. It can be produced as a complete autologous scaffold. It is moldable and degradation is controllable by the use of aprotinin.     

Human adipose tissue as a source of cells with angiogenic potential

Endothelial cells (ECs) are involved in the process of angiogenesis, the outgrowth of new vessels from preexisting blood vessels. If available in sufficiently large numbers, ECs could be used therapeutically to establish blood flow through in vitro engineered tissues and tissues suffering from severe ischemia. Adipose tissue (AT) is an easily available source of large number of autologous ECs. Here we describe the isolation, in vitro expansion, and characterization of human AT derived ECs (AT-ECs). AT-ECs proliferated rapidly through 15-20 population doublings. The cultured cells showed cobblestone morphology and expressed EC markers including CD31, CD144, eNOS, CD309, CD105, von Willebrand factor, CD146, CD54, and CD102. They bound Ulex europaeus agglutinin I lectin and took up DiI-Ac-LDL. The AT-ECs formed capillary-like tubes in Matrigel in vitro and formed functional blood vessels in Matrigel following subcutaneous injection into immunodeficient mice. In conclusion, AT-ECs reach clinically significant cell numbers after few population doublings and are easily accessible from autologous AT, which also contains mesenchymal stem cells/pericytes. Thus, AT yields two cell populations that may be used together in the treatment of tissue ischemia and in clinical applications of tissue engineering.     

Human mesenchymal stem cell implantation and collagen modification as a tool for tissue engineering

Tissue engineering, in all its aspects, is a focus of increasing interest. Optimum vascularization has to be ensured ahead of transferring experimental designs to clinical applications. Our group searched for matrix modifications and cell sources liable to increase vessel formation in engineered tissue. Notwithstanding the ethical issues, adult human mesenchymal stem cells (hMSC) seemed to offer as unlimited a possibility of proliferation and differentiation as embryonic stem cells. These series of experiments focused on the differentiation capability of adult stem cells and organisation in modified collagen sponges. Adult mesenchymal stem cells were cultured to the second generation. Two different groups were formed: Group A cells remained in medium without additional growth factors. Group B cells were cultured in angiogenic differentiation medium. On day 21, the cells of both groups were detached and implanted into different collagen sponges: unmodified collagen sponges and EDC/NHS [1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS)] cross-linked and heparinized collagen scaffolds. Twenty-one days later, the sponges were prepared for the histological evaluation. After 3 weeks in culture, the cells of group B showed phenotypic endothelial cell characteristics and expressed von-Willebrand factor. Enhanced proliferation and invasion on cross-linked and heparinised matrices were obtained with both cell types (A and B). In addition, the stem cell derived endothelial cells formed organised structures throughout the scaffolds. Obtained data revealed the differentiation capability of adult MSC into endothelial cells and organisation in modified collagen matrices. This approach might lead to optimised vascular incorporation in tissue replacement.M. Markowicz, A. Heitland have contributed equally in this work.